Project Summary/Abstract Antibiotic resistant Gram-negative infections pose a serious threat to human health. The outer membrane of Gram-negative bacteria is a unique structure essential for survival; it also functions as a physical barrier to block entry of many classes of antibiotics and thereby render them ineffective. This research is directed towards understanding the structure and function of two multi-protein machines responsible for the biogenesis of two major components of the outer membrane, lipopolysaccharide (LPS) and outer membrane proteins (OMPs). To understand the protein-protein interactions within each machine and their molecular structures, biochemical and structural studies will be undertaken. To dissect the functions of the individual components of these machines, intermediates in transport and assembly of LPS and OMPs will be characterized structurally, biochemically, and in cells. A better understanding of the protein machinery and the processes in which they are involved may lead to the discovery of inhibitors that could ultimately be developed to treat Gram-negative infections.